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jcb Home » 2009 Archive » 5 October » 187 (1): 61
Glyburide inhibits the Cryopyrin/Nalp3 inflammasome
Mohamed Lamkanfi, James L. Mueller, Alberto C. Vitari, Shahram Misaghi, Anna Fedorova, Kurt Deshayes, Wyne P. Lee, Hal M. Hoffman, Vishva M. Dixit
Mohamed Lamkanfi
James L. Mueller
Alberto C. Vitari
Shahram Misaghi
Anna Fedorova
Kurt Deshayes
Wyne P. Lee
Hal M. Hoffman
Vishva M. Dixit
DOI: 10.1083/jcb.200903124 | Published October 5, 2009
Inflammasomes activate caspase-1 for processing and secretion of the cytokines interleukin-1β (IL-1β) and IL-18. Cryopyrin/NALP3/NLRP3 is an essential component of inflammasomes triggered by microbial ligands, danger-associated molecular patterns (DAMPs), and crystals. Inappropriate Cryopyrin activity has been incriminated in the pathogenesis of gouty arthritis, Alzheimer's, and silicosis. Therefore, inhibitors of the Nalp3 inflammasome offer considerable therapeutic promise. In this study, we show that the type 2 diabetes drug glyburide prevented activation of the Cryopyrin inflammasome. Glyburide's cyclohexylurea group, which binds to adenosine triphosphatase (ATP)–sensitive K+ (KATP) channels for insulin secretion, is dispensable for inflammasome inhibition. Macrophages lacking KATP subunits or ATP-binding cassette transporters also activate the Cryopyrin inflammasome normally. Glyburide analogues inhibit ATP- but not hypothermia-induced IL-1β secretion from human monocytes expressing familial cold-associated autoinflammatory syndrome–associated Cryopyrin mutations, thus suggesting that inhibition occurs upstream of Cryopyrin. Concurrent with the role of Cryopyrin in endotoxemia, glyburide significantly delays lipopolysaccharide-induced lethality in mice. Therefore, glyburide is the first identified compound to prevent Cryopyrin activation and microbial ligand-, DAMP-, and crystal-induced IL-1β secretion.
Glyburide is the most widely used sulfonylurea drug for the treatment of type 2 diabetes in the United States (Riddle, 2003). The drug works by inhibiting ATP-sensitive K+ (KATP) channels in pancreatic β cells (Ashcroft, 2005). KATP channels are octameric complexes of four Kir6.x (Kir6.1 or Kir6.2) and four sulfonylurea receptor (SUR; SUR1 or SUR2) subunits (Clement et al., 1997). The SUR subunits belong to the ATP-binding cassette (ABC) transporter family (Aguilar-Bryan et al., 1995) and function as a regulatory subunit, endowing the Kir6.x channel with sensitivity to inhibition by sulfonylureas such as glyburide and glipizide (Ashcroft, 2005). In addition to KATP channels, the ABC transporter ABCA1 was proposed as a putative glyburide target (Hamon et al., 1997). Glyburide's pharmacological properties are summarized in Fig. S1 A.
The cystein protease caspase-1 mediates the proteolytic maturation of the cytokines interleukin-1β (IL-1β) and IL-18 after its recruitment in protein complexes termed inflammasomes (Lamkanfi and Dixit, 2009). Cryopyrin/NALP3/NLRP3 is an essential component of inflammasomes triggered by pathogen-associated molecular patterns (PAMPs), danger-associated molecular patterns (DAMPs), and crystalline substances (Kanneganti et al., 2006, 2007; Mariathasan et al., 2006; Sutterwala et al., 2006; Lamkanfi and Dixit, 2009). Inappropriate Cryopyrin activity has been incriminated in the pathogenesis of several diseases, including gouty arthritis, Alzheimer's, and silicosis (Martinon et al., 2006; Cassel et al., 2008; Dostert et al., 2008; Halle et al., 2008; Hornung et al., 2008), so inhibitors of the Cryopyrin inflammasome offer considerable therapeutic promise.
In this study, we show that glyburide prevented activation of the Cryopyrin inflammasome by a variety of stimuli. Concurrent with the role of Cryopyrin in endotoxemia, glyburide delayed lipopolysaccharide (LPS)-induced lethality in mice. Therefore, glyburide is the first compound identified to act upstream of Cryopyrin to prevent PAMP-, DAMP-, and crystal-induced IL-1β secretion.
Glyburide inhibits LPS+ATP-induced caspase-1 activation, IL-1β secretion, and macrophage death
Glyburide prevents LPS+ATP-induced secretion of IL-1β from human and murine macrophages (Hamon et al., 1997; Laliberte et al., 1999; Perregaux et al., 2001) and from murine Schwann cells (Marty et al., 2005). To determine whether caspase-1 activation is impaired by glyburide, LPS-primed bone marrow–derived macrophages (BMDMs) were incubated with glyburide for 15 min before ATP was added for another 30 min. In contrast to the related sulfonylurea glipizide, glyburide inhibited caspase-1 processing in a dose-dependent fashion (Fig. 1 A), and this prevented secretion of the caspase-1–dependent cytokines IL-1β (Fig. 1 B) and IL-18 (Fig. 1 C). Secretion of IL-6 (Fig. 1 D) and TNF (Fig. 1 E) was not impaired by glyburide, ruling out a general defect in macrophage responsiveness. Inhibition was evident up to 3 h post-ATP (Fig. S1 B), indicating that glyburide did not merely delay caspase-1 activation.
Glyburide inhibits LPS+ATP-induced caspase-1 activation, secretion of IL-1β and IL-18, and macrophage cell death. (A–E) LPS-primed BMDMs were treated with glyburide, glipizide, or DMSO for 15 min before 5 mM ATP was added for 30 min. Cell extracts were immunoblotted for caspase-1 (A), and culture supernatants were analyzed for secreted IL-1β (B), IL-18 (C), IL-6 (D), and TNF (E). Black arrowheads indicate procaspase-1, and white arrowheads mark the p20 subunit. (F) BMDMs were incubated with 200 µM glyburide, 200 µM glipizide, 200 µM DMSO, or 50 µM calmidazolium for 2 h before brightfield photographs were taken. (G) LPS-primed BMDMs were treated with 200 µM glyburide, glipizide, or DMSO for 15 min followed by 5 mM ATP for the indicated durations. Membrane damage was measured using Live/Dead assay. Bars, 20 µm. (H) BMDMs were left untreated (CTRL), stimulated with 10 µg/ml LPS for 3 h, treated with 5 mM ATP for 1 h, or treated with LPS and ATP. Membrane damage was measured using Live/Dead assay. (I) LPS-primed BMDMs from wild-type (WT), P2X7−/−, Cryopyrin−/−, and caspase-1−/− mice were treated with 5 mM ATP for the indicated durations. Membrane damage was measured with Live/Dead assay. Cytokine and cell death data represent the mean ± SD of triplicate samples from a single experiment, and all results are representative of at least three independent experiments.
Significantly, BMDMs cultured for 3 h in glyburide, glipizide, or DMSO looked morphologically normal (Fig. 1 F) and displayed no significant membrane damage (Fig. S1 C). As a positive control, macrophage death was induced with the calmodulin inhibitor calmidazolium (Fig. 1 F and Fig. S1 C). Inhibition of caspase-1 activation by glyburide was reversible because caspase-1 was activated if glyburide was removed from the culture medium before ATP addition (Fig. S1 D). Glyburide also blocked the rapid, caspase-1–dependent cell death that occurs when BMDMs are treated with LPS and ATP (Fig. 1, G–I). As expected, glipizide and DMSO did not prevent this death. Of note, neither LPS nor ATP alone affected BMDM viability (Fig. 1 H). Like caspase-1−/− BMDMs, cells lacking the P2X7 receptor or Cryopyrin were not killed by LPS+ATP (Fig. 1 I). These results demonstrate that the Cryopyrin inflammasome is essential for LPS+ATP-induced macrophage death and that glyburide inhibits LPS+ATP-induced caspase-1 activation, IL-1β secretion, and macrophage death.
Glyburide's sulfonyl and benzamido groups are required for optimal inhibition of the Cryopyrin inflammasome
Glyburide inhibits KATP channels on pancreatic β cells. Its cyclohexylurea group is necessary for high affinity binding to SUR1 (Meyer et al., 1999), which is the SUR subunit of these KATP channels. Interestingly, the cyclohexylurea group (Fig. 2 A, compound G1) was dispensable for inhibition of LPS+ATP-induced caspase-1 activation (Fig. 2 B) and IL-1β secretion (Fig. 2 C). As expected, glyburide and compound G1 did not affect IL-6 secretion (Fig. 2 D). Glipizide also contains a cyclohexylurea group (Fig. 2 A) and inhibits SUR1-containing KATP channels, but it failed to inhibit caspase-1 activation (Fig. 2 B). These data suggest that inhibition of the Cryopyrin inflammasome is independent of SUR1-containing KATP channels.
Glyburide's cyclohexylurea moiety is dispensable for inflammasome inhibition. (A) Chemical structure of glyburide, glipizide, and glyburide-derived analogues G1–G4. (B–G) LPS-primed BMDMs were treated with glyburide, glipizide, or analogues G1–G4 for 15 min before 5 mM ATP was added for 30 min. Cell extracts were immunoblotted for caspase-1 (B and E), and culture supernatants were analyzed for secreted IL-1β (C and F) and IL-6 (D and G). Black arrowheads indicate procaspase-1, and white arrowheads mark the p20 subunit. Cytokine data represent the mean ± SD of triplicate samples from a single experiment, and all results are representative of at least three independent experiments.
Additional structure activity experiments demonstrated that both the benzamido and sulfonyl groups of glyburide (Fig. 2 A, compounds G2–G4) were required for optimal inhibition. Analogue G2 comprising only the benzamido group (Fig. 2 A, compound G2) inhibited LPS+ATP-induced caspase-1 activation (Fig. 2 E) and IL-1β secretion (Fig. 2 F), albeit less effectively than compound G1. Thus, the benzamido group contributes but is not sufficient for optimal inhibition. Indeed, a G1 analogue lacking the benzamido group (Fig. 2 A, compound G3) did not affect caspase-1 activation and IL-1β secretion (Fig. 2, E and F). In addition to the benzamido group, the sulfonyl moiety is required for optimal inhibition, as deletion of the sulfonyl group from compound G1 (Fig. 2 A, compound G4) was less effective at inhibiting caspase-1 activation and IL-1β secretion (Fig. 2, E and F). Like glyburide, compounds G1–G4 permitted normal IL-6 secretion (Fig. 2 G), demonstrating the specificity of these results.
Glyburide inhibits the Cryopyrin inflammasome downstream of the P2X7 receptor
Like ATP, the cation ionophore nigericin engages the Cryopyrin inflammasome (Mariathasan et al., 2006), but nigericin signals independently of the P2X7 receptor (Fig. S1, E–G; Solle et al., 2001). Glyburide potently blocked LPS+nigericin-induced caspase-1 activation (Fig. 3 A). As expected, glipizide and DMSO vehicle did not prevent LPS+nigericin-induced caspase-1 activation. Notably, substituting lipid A, lipoteichoic acid, peptidoglycan, or Pam3-CSK4 for LPS did not prevent glyburide from inhibiting caspase-1 activation (Fig. 3 B).
Glyburide inhibits PAMP-, DAMP-, and crystal-induced activation of the Cryopyrin inflammasome. (A) LPS-primed BMDMs were incubated with 200 µM glyburide, glipizide, or DMSO for 15 min before 20 µM nigericin was added for 30 min. Cell extracts were immunoblotted for caspase-1. (B) BMDMs were stimulated with the indicated PAMPs for 3 h, incubated with 200 µM glyburide for 15 min, and stimulated with 20 µM nigericin for 30 min. Cell extracts were immunoblotted for caspase-1. (C and D) LPS-primed BMDMs were incubated with 200 µM glyburide or DMSO for 15 min before 500 µg/ml silica or1 mM of the lysosomotrophic peptide H-LL-OMe was added for 3 h. Cell extracts were immunoblotted for caspase-1 (C), and culture supernatants were analyzed for secreted IL-1β (D). (E) LPS-primed BMDMs were left untreated or incubated with 200 µM glyburide or 10 µM KN-62 for 15 min. 2 µM YoPro-1 was subsequently added, and its uptake was visualized before and after a 5-min ATP pulse. Bars, 50 µm. (F) BMDMs were left untreated (CTRL), transfected with DOTAP alone for 4 h, stimulated with 10 µg/ml LPS for 3 h and subsequently with 5 mM ATP (LPS+ATP), or transfected with DOTAP and 30 µg/ml LPS for 4 h (DOTAP+LPS) in the presence or absence of 200 µM glyburide, glipizide, or DMSO. Cell extracts were immunoblotted for caspase-1. (G) Overview and mapping of inflammasome-activating stimuli and upstream signaling pathways. Black arrowheads indicate procaspase-1, and white arrowheads mark the p20 subunit. Cytokine data represent the mean ± SD of triplicate samples from a single experiment, and all results are representative of at least three independent experiments.
In agreement with previous studies (Cassel et al., 2008; Dostert et al., 2008; Hornung et al., 2008), silica and the lysosomotrophic molecule H-LL-OMe also promoted Cryopyrin-dependent caspase-1 activation and IL-1β secretion in LPS-primed BMDMs (Fig. S2, A and B). Caspase-1 activation and IL-1β secretion in response to these stimuli were independent of the P2X7 receptor (Fig. S2, C and D) but required TLR4 signaling (Fig. S2 E). Glyburide effectively blocked caspase-1 activation and IL-1β secretion by these stimuli too (Fig. 3, C and D). These data suggest that glyburide targets a signaling component downstream of the P2X7 receptor. We first considered the hemichannel pannexin-1, which was proposed to signal both LPS+ATP- and LPS+nigericin-induced caspase-1 activation (Pelegrin and Surprenant, 2006, 2007; Kanneganti et al., 2007). Pannexin-1 forms a nonselective pore for molecules <1 kD, such as the fluorescent dye YoPro-1, within seconds to minutes of P2X7 receptor stimulation (Pelegrin and Surprenant, 2006, 2007; Locovei et al., 2007). Unlike the P2X7 receptor inhibitor KN-62, glyburide did not prevent ATP-induced YoPro-1 uptake into LPS-primed macrophages (Fig. 3 E), suggesting that pannexin-1 is not inhibited by glyburide. Concurrently, glyburide inhibited caspase-1 activation by LPS+DOTAP (Fig. 3 F), a stimulus that engages the Cryopyrin inflammasome independently of the P2X7 receptor and pannexin-1 (Kanneganti et al., 2007). Collectively, these results indicate that glyburide inhibits engagement of the Cryopyrin inflammasome by diverse stimuli and that inhibition occurs downstream of the P2X7 receptor (Fig. 3 G).
KATP channels and ABC transporters are dispensable for activation of the Cryopyrin inflammasome
The structure–activity relationship experiments (Fig. 2) suggest that glyburide inhibits the Cryopyrin inflammasome independently of SUR1-containing KATP channels. Certain tissues such as heart and muscle express SUR2 instead of SUR1 KATP channels. However, SUR2−/− BMDMs activated caspase-1 and secreted normal amounts of IL-1β in response to LPS+ATP and LPS+nigericin (Fig. 4, A and B). In addition, BMDMs from Kir6.1−/− and Kir6.2−/− mice also demonstrated normal LPS+ATP- and LPS+nigericin-induced caspase-1 activation (Fig. 4 C) and IL-1β secretion (Fig. 4 D). These results show that KATP channels are not required for activation of the Cryopyrin inflammasome.
KATP channels and ABC transporters are dispensable for activation of the Cryopyrin inflammasome. (A–E) BMDMs from wild-type (WT) and SUR2−/− (A and B), Kir6.1−/− and Kir6.2−/− (C and D), or abca1−/−, abcg1−/−, and abca1−/−/abcg1−/− (abca1/g1−/−) mice (E) were left untreated (CTRL), stimulated with 10 µg/ml LPS for 3 h, treated with 5 mM ATP or 20 µM nigericin for 30 min, or stimulated with LPS and treated with ATP (LPS+ATP) or nigericin (LPS+nigericin). Cell extracts were immunoblotted for caspase-1 (A, C, and E), and culture supernatants were analyzed for secreted IL-1β (B and D). (F–H), LPS-primed BMDMs from abca1−/−/abcg1−/− (F), Kir6.1−/− (G), and Kir6.2−/− (H) mice were treated with 200 µM glyburide or DMSO for 15 min before 5 mM ATP or 20 µM nigericin was added for an additional 30 min. Cell extracts were immunoblotted for caspase-1. Black arrowheads indicate procaspase-1, and white arrowheads mark the p20 subunit. Cytokine data represent the mean ± SD of triplicate samples from a single experiment, and all results are representative of three independent experiments.
Previous studies also suggested that glyburide and the ABC transporter inhibitor DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium) target ABCA1 to inhibit LPS+ATP-induced IL-1β secretion (Hamon et al., 1997; Marty et al., 2005). We confirmed that DIDS inhibited LPS+ATP-induced IL-1β secretion in a dose-dependent manner (Fig. S3 A). However, DIDS also inhibited P2X7 receptor-mediated currents (Ma et al., 2009) and caspase-1 activation (Fig. S3 B), suggesting that the drug prevents IL-1β secretion by directly antagonizing the P2X7 receptor rather than ABCA1. Concurrently, BMDMs from mice lacking ABCA1 and/or ABCG1 activated caspase-1 normally in response to LPS+ATP and LPS+nigericin (Fig. 4 E). Caspase-1 activation also was normal in macrophages lacking the glyburide receptor cystic fibrosis transmembrane conductance regulator (unpublished data).
To complement these experiments, we analyzed whether glyburide prevented caspase-1 activation in macrophages lacking ABC transporters or KATP channels. As in wild-type macrophages (Fig. 1 A), glyburide abolished LPS+ATP- and LPS+nigericin-induced caspase-1 activation in macrophages lacking ABCA1 and ABCG1 (Fig. 4 F) or the KATP channel subunits Kir6.1 (Fig. 4 G) and Kir6.2 (Fig. 4 H). In summary, these experiments indicate that known targets of glyburide are dispensable for activation of the Cryopyrin inflammasome.
Inflammasome inhibition occurs upstream of Cryopyrin
Ipaf rather than Cryopyrin is critical for inflammasome assembly and caspase-1 activation by Salmonella typhimurium infection (Mariathasan et al., 2004). Interestingly, caspase-1 activation in S. typhimurium–infected BMDMs was not affected by glyburide (Fig. 5 A). Furthermore, anthrax lethal toxin, which engages the NALP1b inflammasome (Boyden and Dietrich, 2006), also activated caspase-1 normally in the presence of glyburide (Fig. 5 B). Concurrently, glyburide did not affect caspase-1 activity in vitro (Fig. 5 C). These data suggest that glyburide works upstream of caspase-1 and ASC, the shared adaptor in the Cryopyrin, Ipaf, and NALP1b inflammasomes (Fig. 3 G).
Glyburide inhibits the inflammasome upstream of Cryopyrin. (A and B) BMDMs from C57BL/6 (A) or BALB/c mice (B) were stimulated with 10 µg/ml LPS for 3 h and treated with 5 mM ATP for 30 min (LPS+ATP), infected with S. typhimurium for 4 h, or treated with 10 µg/ml anthrax lethal toxin (PA+LF) for 4 h in the presence or absence of 200 µM glyburide. Cell extracts were immunoblotted for caspase-1. Black arrowheads indicate procaspase-1, and white arrowheads mark the p20 subunit. (C) In vitro enzymatic activity of 1 IU mouse caspase-1 incubated with glyburide, glipizide, or the caspase-1 inhibitor Ac-YVAD-cmk. Data represent the mean ± SD of triplicate samples from one out of three independent experiments. (D and E) ATPase activity of purified recombinant Cryopyrin and control eluates in the presence of 100 µM glyburide, compound G1, or glipizide (D). (E) ATPase activity quantified by phosphoimaging. Data represent the mean ± SD of triplicate samples from one out of three independent experiments. (F) Adherent monocytes from FCAS patients (n = 3) were stimulated with 100 ng/ml LPS (4 h) and treated with 5 mM ATP (30 min) in the presence of vehicle controls or 100 µM compound G1 or glipizide before IL-1β levels in culture supernatants were determined. Data represent the mean ± SD of triplicate samples. (G) Adherent monocytes from FCAS patients (n = 3) were cultured at 32°C for 12 h in the presence of vehicle controls or 100 µM compound G1 or glipizide. IL-1β levels were determined in culture supernatants. Data represent the mean ± SD of triplicate samples. (H) Protection against LPS-induced lethality in C57BL/6 mice (n = 10) injected intraperitoneally bid daily with 500 mg/kg glyburide or formulation vehicle. Results are representative of two independent experiments.
We next studied the affect of glyburide and compound G1 on the ATPase activity of Cryopyrin, which is required for caspase-1 activation and IL-1β secretion (Duncan et al., 2007). Neither glyburide nor compound G1 reduced the ATPase activity of recombinant Cryopyrin (Fig. 5, D and E; and Fig. S3, C and D), placing glyburide action upstream of Cryopyrin. We sought confirmatory evidence using human peripheral blood mononuclear cells (PBMCs) from individuals afflicted with familial cold-associated autoinflammatory syndrome (FCAS), a disorder resulting from a temperature-sensitive (L353P) mutation within Cryopyrin (Hoffman et al., 2001). Similar to LPS+ATP at 37°C, cold-induced conformational changes in FCAS monocytes are presumed to assemble the Cryopyrin inflammasome at the permissive temperature of 32°C. It is worth noting that FCAS monocytes secreted a small amount of IL-1β at 37°C in response to either ATP or LPS alone (Fig. S3 E), suggesting that FCAS monocytes are hyperresponsive. Compound G1 inhibited LPS+ATP-induced IL-1β secretion at 37 and 32°C (Fig. 5 F and not depicted) but did not prevent cold-induced IL-1β secretion from FCAS PBMCs at 32°C (Fig. 5 G). Therefore, compound G1 and glyburide likely act upstream of Cryopyrin in inhibiting caspase-1 activation. We also assessed the affect of glyburide on LPS-induced lethality in mice based on the observation that Cryopyrin-deficient mice are resistant to LPS-induced lethality (Mariathasan et al., 2006; Sutterwala et al., 2006). Although 90% of vehicle-treated mice died within 20 h after LPS dosage, all mice in the glyburide-treated group were alive at this time point (Fig. 5 H). Mortality in the glyburide-treated arm was significantly delayed (P < 0.0001), but in line with glyburide's relatively short half-life in vivo (∼1 h; unpublished data), the mice eventually succumbed to endotoxic shock.
Collectively, these results demonstrate that glyburide acts upstream of Cryopyrin and downstream of the P2X7 receptor to block Cryopyrin-dependent inflammasome activation by PAMPs, DAMPs, and crystalline substances. These results may have significant therapeutic ramifications for the treatment of gouty arthritis, silicosis, and Alzheimer's, in which excessive Cryopyrin-dependent IL-1β production was proposed as a major cause of pathology (Cassel et al., 2008; Dostert et al., 2008; Halle et al., 2008; Hornung et al., 2008).
Mice and macrophages
Cryopyrin−/−, caspase-1−/−, Abca1−/−, Abcg1−/−, Abca1−/−Abcg1−/−, Kir6.1−/−, Kir6.2−/−, and SUR2−/− mice have been described previously (Miki et al., 1998, 2002; Schott et al., 2004; Mariathasan et al., 2006; Stoller et al., 2007; Yvan-Charvet et al., 2008). P2X7−/− mice were obtained from Lexicon Pharmaceuticals. Mice were housed in a pathogen-free facility. All experiments were conducted in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Genentech. BMDMs were prepared as described previously (Lamkanfi et al., 2007). In brief, BMDMs were isolated from femurs of 6–12-wk-old mice and were cultured in Iscove's modified Dulbecco's medium containing 10% heat-inactivated FBS, 20% L cell–conditioned medium, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. After 5–7 d of incubation, cells were collected and plated in 6- or 24-well plates in Iscove's modified Dulbecco's medium containing 10% heat-inactivated FBS and 100 mg/ml thymidine and antibiotics. Macrophages were cultured for an additional 24 h before use.
LPS-induced endotoxemia
8-wk-old male C57BL/6 mice (Charles River) were maintained in micro-isolator cages and received food and water ad libitum according to the American Association of Laboratory Animal Care guidelines. Mice were randomly divided into two groups (n = 10/group). The mice were injected intraperitoneally bid daily with 500 mg/kg glyburide or the formulation vehicle (DMSO/10% HP-b-CD [2.5:97.5]) in 360 µl PBS. 4 h after the first dosing, endotoxic shock was induced by intraperitoneal injection of 18 mg/kg LPS (Escherichia coli O111:B4; Sigma-Aldrich). Mice were monitored hourly for survival up to 36 h. Differences in group survival were analyzed with the Kaplan–Meier test. P < 0.05 was considered statistically significant.
The University of California San Diego Human Research Protection Program Committee approved this study, and informed consent was obtained from all subjects. Three related patients with FCAS (L353P mutation) and five normal controls were included. The FCAS patients had a classical clinical presentation, met diagnostic criteria, and were being treated with Rilonacept, an IL-1β inhibitor approved by the Food and Drug Administration, but held their dose for >2 wk before the experiment.
Preparation of human adherent monocytes
Venous blood was drawn into EDTA/Vacutainer tubes (BD) early in the morning when FCAS patients are generally least symptomatic. PBMC fractions were collected and resuspended in serum-free media and transferred to 24-well plates at a concentration of 1 × 106/ml. Cells were incubated for 4 h at 37°C, and nonadherent cells were removed leaving adherent monocytes as previously described (Rosengren et al., 2007).
Bacteria, ligands, and inhibitors
S. enterica serovar typhimurium strain SL1344 was provided by D. Monack (Stanford University, Palo Alto, CA). Single colonies were inoculated into 3 ml of brain-heart infusion medium and grown overnight at 30°C with shaking. Anthrax lethal factor and protective antigen (List Biological Laboratories) were used at 10 µg/ml. Ultrapure LPS, lipid A, lipoteichoic acid, peptidoglycan, and Pam3-CSK4 (InvivoGen) were used at 10 µg/ml. ATP (Roche) was used at 5 mM, and nigericin (Sigma-Aldrich) was used at a final concentration of 20 µM. Glyburide, glipizide, and calmidazolium were obtained from Sigma-Aldrich and used at the indicated concentrations. Compounds G1–G4 were purchased from Ryan Scientific. DOTAP was obtained from Roche and used according to the manufacturer's instructions. Silicia (Min-U-Sil 5) was provided by US Silica, and H-LL-OMe was purchased from Chem-Impex International. Infection and stimulation of BMDMs were performed as described in Figs. 1–5.
Bright-field and fluorescence microscopy were performed in culture media at room temperature under a long-distance Plan Neofluar 20×/0.4 Ph2 Korr objective (Carl Zeiss, Inc.) on a microscope (Axiovert 200M; Carl Zeiss, Inc.) equipped with a charge-coupled device digital camera (Axiocam; Carl Zeiss, Inc.) and AxioVision software (Rel.4.6; Carl Zeiss, Inc.). For dye uptake assays, 2 µM YoPro-1 (Invitrogen) was present 10 min before macrophages were stimulated with ATP for 5 min. Fluorescence signals in digital black and white fluorographs were artificially colored green with Photoshop (Adobe). Macrophage membrane damage was measured with a Live/Dead assay (Invitrogen).
Cytokines, antibodies, and Western blotting
Human and mouse cytokines in culture supernatants were measured by enzyme-linked immunoabsorbent assay (R&D Systems) and Luminex assay. Data were analyzed with Student's t test, and P < 0.05 was considered statistically significant. The caspase-1 antibody used for Western blotting was raised against recombinant mouse caspase-1 and was used as described previously (Lamkanfi et al., 2007). In brief, immunoblots were incubated overnight with the caspase-1 antibody (1:1,000), and a goat anti–rabbit secondary antibody (Jackson ImmunoResearch Laboratories, Inc.) was used to detect proteins by enhanced chemiluminescence (Thermo Fisher Scientific).
In vitro caspase-1 activity assay
In vitro caspase-1 activity was determined by incubating 1 IU recombinant mouse caspase-1 (BioVision) with 50 mM fluorogenic caspase-1 substrate peptide Ac-WEHD-amc in 200 ml CFS buffer (10 mM Hepes, pH 7.4, 220 mM mannitol, 68 mM sucrose, 2 mM NaCl, 2.5 mM KH2PO4, 0.5 mM EGTA, 2 mM MgCl2, 0.5 mM sodium pyruvate, 0.5 mM L-glutamine, and 10 mM DTT). The release of fluorescent 7-amino-4-methylcoumarin in the presence of the indicated concentrations of glyburide, glipizide, or the caspase-1 inhibitor Ac-YVAD-cmk was measured for 30 min at 1-min intervals by fluorometry (excitation at 360 nm and emission at 480 nm) on a Cytofluor (PerSeptive Biosystems), and the maximal rate of increase in fluorescence was calculated (ΔF/min).
ATPase assay
ATP hydrolysis was measured by visualizing the conversion of α-[32P]ATP to α-[32P]ADP using TLC. A total of 5 µl purified Cryopyrin-Flag was incubated with 0.1 µM α-[32P]ATP (3,000 Ci/mmol; PerkinElmer) in a total volume of 20 µl reaction buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1 mM EDTA, and EDTA-free Complete protease inhibitor cocktail tablets) containing 100 µM glyburide, compound G1, glipizide, or vehicle control (DMSO) for 2 h. The reaction was quenched by adding an equal volume of TLC development solvent (1 M formic acid and 0.5 M LiCl). A total of 2 µl reaction mixture was spotted on a polyethyleneimine cellulose TLC plate and developed with 1 M formic acid with 0.5 M LiCl in a TLC chamber. The TLC plate was exposed to x-ray film or used for quantification with a phosphoimager (Typhoon Trio; GE Healthcare).
Online supplemental material
Fig. S1 shows that LPS+ATP-induced caspase-1 activation and macrophage death are glyburide sensitive and, unlike LPS+nigericin, require the P2X7 receptor. Fig. S2 shows that LPS+H-LL-OMe– and LPS+silica-induced caspase-1 activation and IL-1β secretion require Cryopyrin and TLR4 but not the P2X7 receptor. Fig. S3 shows inhibition of caspase-1 activation by DIDS, immunopurification of recombinant Cryopyrin-Flag, and IL-1β secretion from FCAS monocytes. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200903124/DC1.
We thank Andres Paler Martinez, Karen O'Rourke, Xingrong Liu, Yvonne Franke, Matt McGeough, and Carla Pena for technical support, Dr. Elizabeth McNally (University of Chicago, Chicago, IL) for SUR2−/− femurs, Dr. Alan Tall (Columbia University, New York, NY) for Abca1−/−, Abcg1−/−, and Abca1−/−Abcg1−/− femurs, Dr. Andre Terzic (Mayo Clinic, Rochester, MN) for Kir6.1−/− and Kir6.2−/− femurs, and Dr. Peter Vandenabeele (Ghent University, Ghent, Belgium) for caspase-1 antibody.
M. Lamkanfi, S. Misaghi, A.C. Vitari, A. Fedorova, K. Deshayes, W.P. Lee, and V.M. Dixit are employees and/or shareholders of Genentech.
Abbreviations used in this paper:
ATP-binding cassette
BMDM
bone marrow–derived macrophage
danger-associated molecular pattern
FCAS
familial cold-associated autoinflammatory syndrome
interleukin
KATP
ATP-sensitive K+
lipopolysaccharide
pathogen-associated molecular pattern
PBMC
peripheral blood mononuclear cell
sulfonylurea receptor
Submitted: 23 March 2009
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LETTER: Midget hockey players earn respect of coach
This game is more than a game – it's a way of life, it's team camaraderie, it's discipline, says former coach
This year's midget hockey team has seen many changes: six players from last year have graduated or moved on to major midget and juvenile teams.
Sooke Minor Hockey's president has expressed that it is time for a change and a new direction with respect to coaching.
During the last two years as head coach, I had the privilege of taking the boys to the provincial championship in Dawson Creek where we were up against the best in B.C. The players came together and brought home the bronze medal . Last season we made it to the finals, but fell short to a very strong Port Alberni team.
I will miss the games, tournaments and even early morning practices. As a coach, the goal is to develop players as well as strong individuals.
These boys have enriched my life and taught me a lot about myself. I'd like to thank all the parents and players that have supported minor hockey.
This game is more than a game – it's a way of life, it's team camaraderie, it's discipline and, most importantly, it teaches players social skills and work ethic.
All the best for the upcoming season. Work hard boys!
Kevin Berger, Sooke
LETTER: John Phillips Memorial Park a special place


usfws
Blackwater National Wildlife Refuge – Nanticoke Unit Easements
A partnership between the United States Fish and Wildlife Service (USFWS) and Chesapeake Conservancy has led to the permanent protection of 155 acres of land as part of the Nanticoke Unit of Blackwater National Wildlife Refuge. Funding was made possible … Continued
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NFWF Chesapeake Bay Stewardship Fund Grants 2018
In December, the National Fish and Wildlife Foundation (NFWF) and the U.S. Environmental Protection Agency (EPA) announced more than $13.1 million in grants to support the restoration and conservation of the Chesapeake Bay watershed in six U.S. states and the … Continued
agriculture, communities, delaware, District of Columbia, environment, EPA, farming, fish, funding, grants, habitat restoration, maryland, National Fish and Wildlife Foundation, new york, nfwf, ngo, organizations, pennsylvania, runoff, usfws, virginia, water quality, watersheds, west virginia, wildlife
2017 Chesapeake Bay Stewardship Fund Grants
In September, the National Fish and Wildlife Foundation (NFWF) and the U.S. Environmental Protection Agency (EPA) announced more than $12.6 million in grants to support the restoration and conservation of the Chesapeake Bay watershed in six states and the District … Continued
baltimore, EPA, grants, habitat restoration, National Fish and Wildlife Foundation, nfwf, pollution, rivers, runoff, usfws, water quality, watersheds


Former tourist mecca still draws those eager to sip or soak in its medicinal waters
Image: Jennifer Nichols
Water plays a starring role in Mineral Wells' history—and its present, as well.
Visitors to the small city about 50 miles west of Fort Worth can view tantalizing glimpses of its storied past as one of the country's premier spa resorts and explore the area's distant past at the bottom of an ancient sea.
A stroll around downtown gives one a sense of the prosperity that sprang from the earth around the turn of the 20th century. A mineral-rich aquifer tapped by a well driller in the 1880s spurred the establishment of dozens of businesses that catered to thousands of tourists who arrived to drink or bathe in the water, which gained the reputation for curing ailments both physical and mental.
Remnants of that water boom—downtown streets lined with brick buildings that today house a few antique shops and a collection of Victorian-era homes—remain. Looming over them all is the abandoned but still impressive Baker Hotel, a 450-room, 14-story edifice that opened in 1929 and closed in the 1970s. Gazing at the crumbling structure, it's easy to imagine the days when the once-grand hotel hosted political conventions and Hollywood stars.
Just blocks from the hotel is the only remaining commercial source of the town's mineral water, the Famous Mineral Water Company. The company, founded in 1904, ships its Crazy Water to several states. It also has recently begun offering visitors the chance to soak. For $28, you get a bottle of their mineral water in one of four formulations to sip as you settle into a deep bathtub in a quiet room and relax in a warm bath.
Water helped form another city attraction, with a history going back 300 million years. At the Mineral Wells Fossil Park, visitors can pick through sand and rocks at a former city quarry to find the remains of prehistoric creatures that lived there when that area of Texas was covered by a shallow sea.
In about an hour's worth of casual hunting, I collected a pocketful of interesting specimens, including mollusk shells and bits of crinoid, a type of seabed-dwelling animal. There is no charge to look for or to take home fossils for personal use, but commercial collecting is forbidden. To get to the park, follow the partially paved Indian Creek Road, just west of town off U.S. 180. Take plenty of water. If I went again, I'd bring a garden rake for easier sifting.
Water plays a big role in a more colorful city attraction, this one about 3 miles east of town off U.S. 180. The Clark Gardens Botanical Park is a lush oasis in the middle of mesquite-dotted ranchland. Its 35 acres of carefully tended gardens, which began as the private garden of founders Max and Billie Clark, include hundreds of species of trees and plants in a variety of settings. Lakes, fountains, pavilions and statuary invite visitors to linger. One of my favorite features was an extensive model train layout that incorporated scenery and structures made of natural materials.
After all that traipsing, a rest was in order. The Silk Stocking Row bed-and-breakfast, housed in a turn-of-the-century mansion a couple of blocks from the Famous Mineral Water Company, provided a comfortable and peaceful respite. Hosts Wilene and Chuck Manis offer five themed rooms and a hearty breakfast to start the day.
Whether it's history, natural beauty or a peaceful day off, Mineral Wells lets you soak it all in.
Kevin Hargis is a staff copy editor.
This article appeared in the October 2013 issue


Web site takes you way back in Internet history
JOHN COOK, SEATTLE POST-INTELLIGENCER REPORTER
Ever dream of floating back in time to the good old days of the Internet, when Amazon.com was just a tiny little online bookstore and WebCrawler was the best search engine around?
Well, if you are feeling a bit nostalgic for 1996, it may be time to take a magical ride on The Wayback Machine.
A digital time travel device of sorts, The Wayback Machine allows history buffs, journalists, social anthropologists, researchers and curious Web surfers to scroll the annals of Internet history by pulling up forgotten Web pages.
Want to see Google's first Web site?
Check out Microsoft's home page from October 1996?
Or see Al Gore's Web site from the 2000 election?
It's all right at your fingertips in The Wayback Machine, web.archive.org/
In fact, The Wayback Machine is the largest known database in the world, with about 10 billion archived Web pages taking up 100 terabytes of data. That's enough information to put stresses and strains on the 300 computer servers powering the digital archive.
Brewster Kahle and Bruce Gilliat -- founders of the Amazon.com subsidiary Alexa Internet -- created the concept behind The Wayback Machine in 1996, compiling millions of Web sites for their Internet navigation service. Instead of discarding the old Web properties like other search engines, Kahle and Gilliat stored them in a digital library for a non-profit San Francisco organization known as The Internet Archive.
The digital library grew and grew and grew. But few people knew about it.
Information was kept on digital tape for five years, with Kahle occasionally allowing researchers and scientists to tap into the clunky database.
But when the archive hit its five-year anniversary earlier this year, Kahle and the rest of the team decided to open it to the public. In a ceremony last week at the University of California-Berkeley, The Wayback Machine was unveiled.
Now, for the first time in five years, all of the information is available free to the public.
For those who followed the birth of the Internet, The Wayback Machine is kind of like opening King Tut's tomb for the first time. It's a virtual treasure trove of information.
So far, it has been a smashing success, with more than 1 million visitors per day.
"People love to see their old Web sites. They are just thrilled to see the old pictures that they thought were long gone," Kahle said. "And they are psyched about becoming a part of history."
With a good memory of Web-site addresses and a quick click of the "Take Me Back!" button, one can spend hours exploring the darkest corners of cyberspace.
I checked out one of Amazon.com's early home pages, a primitive site that showed a grainy looking logo that boasted "Earth's biggest bookstore." Comparing it with Amazon.com's site today -- with its multiple tabs, consumer reviews and colorful graphics -- shows just how far e-commerce has progressed in five years.
I also explored Web sites of Seattle companies that are no longer with us such as HomeGrocer.com, Mercata, Rivals.com and FizzyLab. After typing in the names and waiting a few seconds, they all popped up.
In many cases, the links within the Web sites even worked -- allowing deeper exploration of news releases, management teams and investors.
Hidden gems are found all over.
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For example, check out this message from HomeGrocer.com dated Nov. 16, 1999:
"What if you didn't have to go to the grocery store? What if a truck drove up to your door, and a nice driver put the groceries on your kitchen counter? What if the groceries didn't cost more, and they were as good as or better than what you normally buy? Too good to be true? Well, we'll give you a 100 percent satisfaction money-back guarantee."
But The Wayback Machine doesn't always work. When I typed in MyLackey.com, the Seattle online errand service that went out of business last year, it returned 89 different Web pages from Jan. 25, 1998, to May 31, 2001. But none of them produced the familiar orange and blue Web site with Mr. Lackey wearing his top hat. A message appeared saying the Web site I requested was not in the archive, but it would be added to a list.
Still, at 10 billion Web pages -- a database that Kahle says is bigger than those operated by Wal-Mart and American Express -- you can find some very interesting things.
In addition to the search function, The Wayback Machine has four special collections: Election 2000, United States Government, Web Pioneers and September 11th.
Still in its early stages, Kahle says he hopes The Wayback Machine can be a useful research tool.
Kahle recalls a Wall Street Journal reporter who contacted him a few years ago to find out exactly when Microsoft changed strategies and decided to make the giant leap into the Internet business.
"He wanted the evidence," Kahle remembered. "But I couldn't provide it because we didn't have a Wayback Machine."
Now, he said, a reporter or researcher could jump on site, type in the Microsoft URL and search through early versions of the company's Web site for evidence.
And that, Kahle said, is a valuable tool.
"Society needs objective views of the past," Kahle said. "The Web ... is becoming an important part of our information ecology."
The Wayback Machine, which takes its name from an imaginary device in the 1960s cartoon "The Adventures of Rocky and Bullwinkle," is backed by AT&T Research, Compaq Computer, Xerox PARC, the Library of Congress, the National Science Foundation and others.
JOHN COOK


COVID-19 Cases Quadruple At Federal Migrant Detention Center In Arizona
By James E. Garcia Jun 18, 2020
A worker at a Honeywell International Inc. factory works on N95 masks May 5, 2020, in Phoenix, Arizona. (Photo by BRENDAN SMIALOWSKI/AFP via Getty Images)
PHOENIX – The number of migrant detainees testing positive at a federal migrant detention center in Arizona has jumped more than 400 percent in the past five days, accounting for 50 percent of the growth in coronavirus cases among detained migrants nationwide.
A spokeswoman for Immigration and Customs Enforcement (ICE) told the Arizona Mirror that the increase is due to more testing. "As a result of these increased efforts, more cases have been identified," said ICE spokesman Yasmeen Pitts O'keefe, who added that "the Eloy Detention Center has also restricted intake for all new admissions to the facility to further protect those in custody."
Arizona is among several states in the Southwest that reported record increases this week in the number of COVID-19 cases and hospitalizations. On Tuesday, the state reported 2,392 new infections. It's been a month since Arizona's stay-at-home order expired.
In a recent press conference, Governor Doug Ducey told reporters "this virus is something we need to learn to live with." But a growing chorus of state leaders, including Congressman Ruben Gallego, have called on Ducey to mandate that Arizonans wear masks to slow the spread of the disease.
In a lawsuit filed earlier this month, attorneys said detainees at the Eloy facility are "trapped in what are essentially tinderboxes on the verge of explosion."
Man Shot In Albuquerque As Protesters Try To Tear Down Statue Of Spanish Conquistador
Stephen Ray Baca, a 31-year-old former city council candidate was arrested Tuesday and charged with aggravated assault with a deadly weapon, after allegedly shooting a man who was with protesters trying to tear down the statue of a Spanish conquistador.
Cellphone video "showed protesters hitting Baca with a skateboard, punching him and chasing him before he opened fire," according to the Associated Press.
Albuquerque Mayor Tim Keller said at a press conference that a separate video taken earlier in the day showed Baca "agitating at the protest well before the shooting" and throwing a woman to the ground. The woman was also pepper sprayed by Baca.
The statue was being guarded by a local militia group called "The New Mexico Civil Guard." Police have not confirmed if Baca was a member of the group. Keller told reporters the New Mexico State Police are investigating the incident, adding that the statue would be removed and placed in storage for the time being.
KRQE News 13 reported that multiple protesters had called police to the scene "as tensions rose" before the shooting, "but police did not arrive until after the protester was shot."
According to the AP, the man who was shot is named Scott Williams. He suffered several gunshot wounds to his torso. Authorities said he was in critical but stable condition at a local hospital.
My statement on the "militia" and violence in Albuquerque tonight: pic.twitter.com/BZISvyau3k
— Michelle Lujan Grisham (@GovMLG) June 16, 2020
In a statement released by Gov. Michelle Lujan Grisham on Twitter, she condemned the violence, saying she was "horrified and disgusted" by the shooting. She criticized the militia group for showing up to "menace the protesters … To menace people of New Mexico with weaponry."
Amid COVID-19 Crisis, Trump Administration Targeting Immigrants On Multiple Fronts
As the nation continues to reel from the deadly effects of the COVID-19 pandemic, The Trump Administration has moved forward with several major policy initiatives aimed at curbing immigration to the United States.
In one of its latest moves, the administration has proposed making extensive changes to asylum law. Critics say "the sweeping restrictions [such as penalizing asylum seekers for not paying taxes or being in the country undocumented for longer than a year] would make it easier for immigration judges to reject asylum requests out of hand."
The goal is to put rules in place that could be enforced long after the pandemic has ended.
Tens of thousands of migrants forced to remain in camps and shelters in Mexico at our southwest border have already been "effectively blocked" from seeking asylum, according to the New York Times, and an executive order has temporarily suspended the "issuance of green cards to many outside the United States."
In April, U.S. Education Secretary Betsy DeVos barred undocumented immigrants, including recipients of Deferred Action for Childhood Arrivals (DACA), from receiving COVID-19 relief funds to help with emergency expenses, such as housing, food or health care. DACA recipients just received the news today that DACA will not be suspended after a decision from the Supreme Court rejected Trump's effort to end the program.
In the coming days, federal officials are expected to announce restrictions to student and work visas, as a follow up to limits to temporary work visas ordered in April. "The White House has made it clear it's considering suspending H-1B visas for high-skilled workers; H-2B visas for seasonal workers and L-1 visas for employees transferring within a company to the U.S," according to the Associated Press.
Supreme Court Declines To Hear White House Challenge To California 'Sanctuary Laws'
The U.S. Supreme Court rejected an appeal to a lower court ruling that upheld California's right to "limit cooperation between state and local law enforcement and federal immigration authorities," according to The Hill.
The Trump administration claimed the legislation interferes with federal immigration law enforcement. California's former Senate President pro Tem Kevin de León, who proposed the law, cheered the justices' decision.
"We already have enough issues with law enforcement being weaponized against the communities that they were intended to protect," de León told the Sacramento Bee. De León was recently elected to the Los Angeles City Council.
Vanguardia America is a free weekly newsletter. To sign up, email Editor and Publisher James E. Garcia at [email protected]
ACLU Condemns Trump Administration's Restriction Of Asylum Process For Migrants At US-Mexico Border
As Latinos Are Among Hardest Hit By Pandemic, Leaders Expect Federal Census Undercount
Amid Recession, Latinos Now Have The Highest Unemployment Rate In The Country


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Germany's Kroos relishing shot at redemption against Dutch, French
AFP October 11, 2018 AFP No Comments
Berlin (AFP) – Toni Kroos says Germany are relishing the chance for redemption following a woeful World Cup campaign as they prepare for crunch Nations League matches away to the Netherlands and France.
"Those games sound good to the ear and we hear the ringing of six points," quipped the Real Madrid midfielder.
"We're motivated, we want two wins."
A goalless draw against France in Munich last month leaves Germany second in Group One of the Nations League, three points behind leaders France.
Having finished bottom of their group in Russia following a shock defeat to South Korea, victories over the Dutch in Amsterdam on Saturday and against the French next Tuesday in Paris would go a long way in helping coach Joachim Loew repair the damage to his hitherto unblemished reputation.
Kroos insists "three games at the World Cup will not change what we've been building for years", although claiming six points over the coming days should help ease the pressure on Loew, who has faced criticism in the German media.
On Thursday, former Germany star Michael Ballack said he was "surprised" the 58-year-old Loew had kept his job after the World Cup debacle.
"Maybe he wanted to take over…," Kroos replied tongue-in-cheek to Ballack's comments as he jumped to Loew's defence ahead of the coach's 168th international in charge on Saturday.
"New stimuli can also come from a coach who has been involved for a long time," insisted Kroos, 28.
"He (Loew) asked himself after the World Cup whether he can still perform to his best.
"I think he deserves the chance and I think we will sort ourselves out with him."
Nevertheless, the away matches in Amsterdam and Paris present a huge challenge.
As assistant coach Marcus Sorg put it "in the Nations League, you can't afford a defeat, then you don't have matters in your own hands any more.
"That is an incredibly high demand."
Manchester City winger Leroy Sane, a surprise omission from the World Cup squad, is set to start on the left flank with Loew's first-choice Marco Reus missing with a knee injury.
Injuries elsewhere have forced Loew to make further changes. Kevin Trapp, Antonio Ruediger, Leon Goretzka, Kai Havertz and Reus have dropped out of the original 23-man squad and been replaced by Emre Can, Serge Gnabry and Bernd Leno.


How U.F.O.s 'Exploded' Into Public View
In the early morning of July 20, 1952, Capt. S.C. "Casey" Pierman was ready for takeoff at Washington National Airport, when a bright light skimmed the horizon and disappeared. He did not think much of it until he was airborne, bound for Detroit,
By Laura M. Holson
and an air traffic controller told him two or three unidentified flying objects were spotted on radar traveling at high speed.
The controller told Captain Pierman to follow them, the pilot told government investigators at the time. Captain Pierman agreed, and headed northwest over West Virginia where he saw as many as seven bluish-white lights that looked "like falling stars without tails," according to a newspaper report.
The sighting of whatever-they-were garnered headlines around the world. And in the decades since, U.F.O.s have become part of the pop culture zeitgeist
MY UFO EXPERIENCE: Eye Witness Recounts 1952 'Flying Saucer' Flap
8 to 12 Saucers (UFOs) Appear Over Washington Again; U.S. Calls in Scientists
Air Force Issues 'Shoot Down Orders' For Flying Saucers (UFOs)!
Mysterious Objects Over Washington Again Today!
MY UFO EXPERIENCE | STORYTIME
UFO Sighting in West Richland, Washington on…
UFO Sighting in Georgia on 1973-05-25 17:00:00 -…
'Oblate Spheroid Space Ships' From Older Planets,…
UFO Sighting in Bothell, Washington on 2019-05-07…
Tagged with: exploded • public


2023: Jega Disagrees With IBB Over Age Limit For President
Olugbenga Ige
Former Chairman of the Independent National Electoral Commission (INEC), Professor Attahiru Jega, has disagreed with the former military president, Ibrahim Babangida on the age bracket of Nigeria's next president.
Recall that in a televised interview, Babangida had said he already had in mind the ideal next president of Nigeria and that the candidate is in his 60s.
The former military dictator said the idea of a good Nigerian leader is someone who has a friend everywhere, must also be well-versed in economics, and be a good politician able to talk to Nigerians.
But in an interview on ARISE NEWS on Monday night, Jega said the next president should be the best choice of the people of Nigeria, adding that age should not matter.
He said: "That is his (Babangida) personal opinion. But as far as I am concerned, the President of Nigeria should be the best choice of the people of Nigeria. Age may not matter.
"As a political scientist, I have read about Nigerian politics, I understand the challenges and I have also had the privilege for five years of relating relatively more loosely with Nigerian politicians, so, I have a clear appreciation of the serious challenges that Nigeria faces particularly, the dangerous trajectory, which many of our politicians are leading this country to and if we all sit and watch and allow this, in no time they would wreck this country."
Speaking on the electoral amendment bill before the National Assembly, Jega said that the bill should not be passed until certain critical elements are addressed.
He noted that if those elements are not attended to, it would create more problems than it intend to solve.
"The whole idea of the electoral amendment is to improve the legal framework. The 2010 Electoral Act as amended is certainly better than the 2006 Electoral Act, which preceded it. But there are still areas that need to be improved on. I have told you one area which since 2011 when I was there. After the election, we discovered how political parties were exploiting the legal framework to undermine the democratic process and INEC could do nothing about it, because the law really arm-strong its capacity to do it. Those are the things you expect that would improve.
"Before the 2019 election, at the last minute, something was done but it was never signed into law until after the election. And now we are expecting before 2023 that we can have a much remarkable; more improved Electoral legal framework. And I'm telling you from what we have seen from the draft bill under consideration by the Senate and yeah, House of Representatives, there are going to be serious remarkable constraints, rather than creating a better framework," he added.
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Developer Creates Holographic Window Using Microsoft's Kinect Sensor
by Microsoft News
@@msftnws
Mar 5, 2015 at 21:36 GMT 6 years ago
Valve's Portal game is known for it's gameplay where portals could be used to teleport between different locations. They are rendered as virtual windows into the connected location with the well-known orange and blue rings around them. A developer has now created a real life portal using Kinect sensors. Watch the video above.
The Kinect V2 is a sensor that can be used to record a 3D view of the world in real-time. It can also track users and can see what their body pose is. This can be used to perform head tracking and reconstruct a camera view into a 3D world as if looking through a virtual window. By using one Kinect for head tracking and another Kinect for reconstructing a 3D view, the virtual window effect of a portal can be created in reality. By using both Kinects for 3D world reconstruction and head tracking a two way portal effect can be achieved.
Read more about it at Channel9.


Springdale Poverty
Springdale borough, Pennsylvania Welfare
Poverty in Springdale borough, Pennsylvania
What is the poverty rate in Springdale borough, Pennsylvania?
The poverty rate in Springdale is 9.5%. One out of every 10.5 residents of Springdale lives in poverty.
How many people in Springdale borough, Pennsylvania live in poverty?
319 of 3,344 Springdale residents reported income levels below the poverty line in the last year.
How does the poverty rate in Springdale compare to the rest of Pennsylvania?
The Poverty Rate across the state of Pennsylvania is 13.1%, meaning Springdale has a significantly lower than average percentage of residents below the poverty line when compared to the rest of Pennsylvania.
Springdale borough, Pennsylvania Poverty Rate By Race
What's the Poverty Rate in Springdale borough, Pennsylvania By Race?
9.0% of White residents of Springdale borough, Pennsylvania live below the poverty line.
The Poverty Rate of white residents in Springdale borough, Pennsylvania is dramatically lower than the national average of 10.3%. 289 of 3,229 white Pennsylvanians live below the poverty line. Approximately 95.7% of the total population of Springdale borough, Pennsylvania are white.
Springdale borough, Pennsylvania Poverty Rate By Sex
What is the poverty rate for men vs women living in Springdale borough, Pennsylvania?
The Poverty Rate for men who live in Springdale borough, Pennsylvania is 4.4%.
The Poverty Rate for women who live in Springdale borough, Pennsylvania is 13.8%.
Poverty Rate Disparity for Male vs Female Residents of Springdale borough, Pennsylvania
Female residents of Springdale borough, Pennsylvania are dramatically(213%) more likely to live in poverty than males.
66 of 1,517 males in Springdale live below the poverty line.
253 of 1,827 females in Springdale live below the poverty line.
Springdale borough, Pennsylvania Poverty Rate By School Enrollment
Understanding where poverty hits hardest in Springdale can be better understood by segmenting poverty by whether or not the resident is in school or not.
Enrolled in School in Springdale borough, Pennsylvania have a Poverty Rate of 12.1%.
Not Enrolled in School in Springdale borough, Pennsylvania have a Poverty Rate of 8.8%.
Poverty for younger ages in Springdale is seen as the most undesirable for the future of the community.
Enrolled in Nursery School in Springdale borough, Pennsylvania have a Poverty Rate of 17.4%.
Enrolled in Elementary School(Grades 1-4) in Springdale borough, Pennsylvania have a Poverty Rate of 0.0%.
Poverty for developing children and young adults in Springdale can have drastic impacts on graduation and college attendance rates.
Enrolled in Middle School(Grade 5-8) in Springdale borough, Pennsylvania have a Poverty Rate of 5.6%.
Enrolled in High School (Grades 9-12) in Springdale borough, Pennsylvania have a Poverty Rate of 26.0%.
Enrolled in Undergraduate School in Springdale borough, Pennsylvania have a Poverty Rate of 4.5%.
Enrolled in Graduate or Professional School in Springdale borough, Pennsylvania have a Poverty Rate of 8.6%.
Springdale borough, Pennsylvania Poverty Rate By Age
What's the Poverty Rate By Age in Springdale borough, Pennsylvania?
Breakdown of Springdale borough, Pennsylvania Poverty Rate By Age
Springdale borough, Pennsylvania Poverty Rate By Place of Birth
What is the poverty rate for residents of Springdale borough, Pennsylvania born US Citizens vs Foreign-Born?
The Poverty Rate for residents of Springdale borough, Pennsylvania who were born citizens of the United States is 9.7%.
The Poverty Rate for residents of Springdale borough, Pennsylvania who were born citizens of a country other than the US is 0.0%.
Poverty Rate for residents of Springdale borough, Pennsylvania born US Citizens vs Foreign-Born
Residents of Springdale borough, Pennsylvania who were born as US Citizens have a higher poverty rate than those who were born citizens of a country outside of the United States.
Of the 3,299 residents of Springdale borough, Pennsylvania who were born citizens of the United States, 319 (9.7%) had income levels below the poverty line within the past year.
Conversely, 0 (0.0%) of 45 residents of Springdale borough, Pennsylvania who were born citizens of a country other than the US reported income levels below the poverty line within the past year.
Poverty Rate in Springdale borough, Pennsylvania
The poverty rate in Springdale is 9.5%.
Springdale is in Allegheny County.